- Email: [email protected]
Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells
Experimental Eye Research 88 (2009) 495–503
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells Alice L. Yu a, Reinhard L. Lorenz b, Christos Haritoglou a, Anselm Kampik a, Ulrich Welge-Lussen a, c, * a
Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336 Munich, Germany Institute for Prophylaxis of Cardiovascular Diseases, Ludwig-Maximilians-University, Pettenkoferstrasse 9, 80336 Munich, Germany c Department of Ophthalmology, Friedrich-Alexander-University, Schwabachanlage 6, 91054 Erlangen, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2008 Accepted in revised form 30 October 2008 Available online 27 November 2008
Age-related macular degeneration (AMD) and artherosclerosis share common characteristics in their pathogenesis. In this study, we investigated the effects of lipoproteins like native (n)-LDL, oxidized (ox)LDL and high-density lipoprotein (HDL) on advanced senescence, extracellular matrix accumulation, cell loss, and transforming growth factor-beta2 (TGF-b2) expression in cultured human retinal pigment epithelial (RPE) cells. Primary human RPE cells were incubated with 10–100 mg/ml n-LDL, ox-LDL, and HDL for 24 h. For determination of advanced senescence, beta-galactosidase staining was used. The induction of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) mRNA was quantified by real-time PCR. Cell loss was investigated by live dead assay. Expression of TGF-b2 was analyzed by real-time PCR and ELISA assays. Ox-LDL accelerated dose-dependently the onset of RPE senescence, whereas LDL and HDL had no effect. LDL and ox-LDL led to induced expression of Fn, Laa1 and Col4a2, whereas HDL had no influence. Incubation of RPE cells with 100 mg/ml ox-LDL induced marked cell death compared to untreated control cells. Expression of TGF-b2 was dose-dependently increased by LDL and ox-LDL. LDL and ox-LDL induced cellular changes in RPE cells in vitro, which may resemble pathogenic events of AMD. These results may provide further information about the effects of LDL and ox-LDL in the human RPE and their potential role in the pathogenesis of AMD. Ó 2008 Published by Elsevier Ltd.
Keywords: lipoprotein retinal pigment epithelium age-related macular degeneration
1. Introduction Age-related macular degeneration (AMD) is the leading cause of visual loss in the elderly in Western societies (Hawkins et al., 1999; Klein et al., 1992; Vingerling et al., 1995a). Until now, the cause of AMD is still unclear. It is suspected that both genetic and environmental factors may play a role in the disease development and progression (Chamberlain et al., 2006; DeWan et al., 2007; Edwards and Malek, 2007; Schmidt et al., 2007). A number of epidemiological studies suggest that AMD shares common risk factors with atherosclerosis (Robman et al., 2004; Vingerling et al., 1995b) like smoking (Mitchell et al., 2002; Smith et al., 2001; Thornton et al., 2005; van Leeuwen et al., 2003), hypertension (Hyman and Neborsky, 2002; Klein et al., 2003, 2004) and elevated cholesterol (Guymer et al., 2005; Mares-Perlman et al., 1995; Tan et al., 2007).
* Corresponding author. Present address: Augenklinik der Friedrich-AlexanderUniversita¨t, Schwabachanlage 6, 91054 Erlangen, Germany. Tel.: þ49 9131 44731; fax: þ49 9131 8534630. E-mail address: [email protected] (U. Welge-Lussen). 0014-4835/$ – see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.exer.2008.10.028
The development of AMD is assumed to start from senescenceassociated RPE changes (Feher et al., 2006). AMD is further characterized by diffuse thickening of Bruch’s membrane due to extracellular matrix (ECM) accumulation such as fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) (An et al., 2006; van der Schaft et al., 1994). Bruch’s membrane represents a specialized vascular intima between the retinal pigment epithelium (RPE) and choriocapillaris (Sivaprasad et al., 2005). Further characteristic AMD changes include primary loss of RPE cells (Roth et al., 2004) and increased levels of transforming growth factor-beta (TGF-b) (Kliffen et al., 1997). The reason for these changes is still unknown. Pathological correlations have been found in atherosclerotic lesions in the intima of large arteries. In atherosclerotic plaque formation, similar events have been observed, such as advanced senescence of endothelial and smooth muscle cells (Al-Shaer et al., 2006; Minamino et al., 2004), diffuse intimal thickening (Newby, 2005), loss of vascular endothelial cells (Stoneman and Bennett, 2004), and increased expression of TGF-b (Cipollone et al., 2004; Zhu et al., 2005). Several lines of evidence suggest that in these atherosclerotic lesions, oxidative modification of low-density lipoprotein (LDL) in the vessel wall is the key event in atherogenesis
496
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
(Schwartz et al., 1991). This critical step is initiated by binding of LDL to the LDL receptor-related protein (LRP), a cell-surface receptor, and subsequent oxidation by 12/15-lipoxygenase (Takahashi et al., 2005). Oxidized low-density lipoprotein (ox-LDL) is the bioreactive variant of LDL and the major contributor for arteriosclerotic changes (Nakajima et al., 2006; Tsimikas, 2006). In atherogenesis, ox-LDL is internalized by activated macrophages via their scavenger receptors like CD36 leading to foam cell formation (Rahaman et al., 2006). In the eye, a broad range of evidence implicates that abnormal age-related accumulation of lipids and cholesterol takes place in the basal laminar deposits of the underlying Bruch’s membrane (Ruberti et al., 2001; Holz et al., 1994; Malek et al., 2003). It has been shown that the spatial distribution of accumulated lipids correlates with the lesions of AMD (Ruberti et al., 2001; Holz et al., 1994). This observation has suggested that lipids may be involved in the pathogenesis of AMD. Furthermore, the high metabolic activity of photoreceptor outer segments and the formation of free radicals are thought to implicate a high local turnover rate of oxidatively modified lipids in RPE (Godley et al., 2002). In previous studies, it has been demonstrated that CD36 scavenger receptors are also highly expressed in RPE cells (Gordiyenko et al., 2004; Ryeom et al., 1996; Yesner et al., 1996). Thus, there is growing evidence that LDL and ox-LDL may contribute to the pathogenesis of AMD (Gordiyenko et al., 2004; Kamei et al., 2007). However, until now, no studies exist to show whether or not LDL and ox-LDL have the potential to induce cellular changes as seen in AMD. The goal of the present study is to investigate the effects of native (n)-LDL and ox-LDL on characteristic RPE changes as found in AMD, such as senescent changes, synthesis of ECM components, cell loss, and expression of TGF-b2 and in cultured human RPE cells. 2. Methods 2.1. Isolation of human RPE cells Ten human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 4–16 h after death. The donors ranged in age from 38 to 63 years. None of the donors had a known history of eye disease. Methods of securing human tissue were humane, included proper consent and approval, complied with the declaration of Helsinki, and was approved by the local ethic committee. Human RPE cells were harvested following the procedure as described previously (Alge et al., 2002; Campochiaro et al., 1986). In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% polyvinylpyrrolidone iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in the sodium chloride solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera using fine forceps. The eyecup was rinsed with Ca2þ- and Mg2þ-free Hank’s balanced salt solution, and treated with 0.25% trypsin (GIBCO, Karlsruhe, Germany) for 1 h at 37 C. The trypsin solution was aspirated and replaced with Dulbecco´s modified eagles medium (DMEM, Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS) (Biochrom). Using a pipette, the media was gently agitated, releasing the RPE into the media by avoiding damage to Bruch’s membrane. 2.2. Lipoprotein preparation and oxidation Lipoproteins were prepared from 80 ml blood from healthy donors by gradient ultracentrifugation (Rubic and Lorenz, 2006; Schulz et al., 1995; Weber et al., 1999). In brief, plasma was carefully
pipetted into four ultracentrifugation tubes (polyallmore centrifuge tubes, Beckmann, Fullerton, CA) and overrun with a density gradient (3 ml d1 ¼1.080 g/ml and 3 ml d2 ¼ 1.050 g/ml of potassium bromide and ethylenediaminetetraacetic acid (EDTA) 0.1% (Titriplex III pa Merck, Germany) in aqua bidest and 2 ml d3 ¼ 1.0 g/ ml EDTA 0.1% in aqua bidest, pH 7.4). Gradient centrifugation was performed in a Beckmann L7-55 ultracentrifuge run at 36,000 rpm and 10 C for 24 h. LDL and HDL subfractions were aspirated from the gradient and stored at 4 C under nitrogen in darkness no longer than 8 h until oxidation. Before oxidation, native LDL was desalted using Econo Pac DG 10 chromatographic columns (Biorad, Munich, Germany). Then, native LDL was oxidized by incubation of EDTA-free LDL with 10 mM CuCl2 in phosphate-buffered saline (PBS) for 16 h at 37 C. The oxidation was monitored at 234 nm in an Uvikon 930 spectrophometer. After 16 h of incubation, 0.24 mM EDTA was added to stop oxidation. Protein content was quantified by a modification of Lowry’s method. Ox-LDL was concentrated with Centriflo Cones (BioRad) (2200 rpm, 20 min, 4 C) and washed twice with PBS buffer. 2.3. Human RPE cell culture The human RPE cell suspension was added to a 50 ml flask (Falcon, Wiesbaden, Germany) containing 20 ml of DMEM supplemented with 20% FCS and maintained at 37 C and 5% carbon dioxide (CO2). Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pan-cytokeratin antibody (Sigma, Deisenhofen, Germany) (Leschey et al., 1990). Cells were tested and found free of contaminating macrophages (anti-CD11, Sigma) and endothelial cells (anti-von Willbrand factor, Sigma). After reaching confluence, primary RPE cells were subcultured and maintained in DMEM supplemented with 10% FCS at 37 C and in CO2. Confluent primary RPE cells of passage 3–5 were used for the experiments. To test the effects of native low-density lipoprotein (n-LDL), oxidized low-density lipoprotein (ox-LDL), and highdensity lipoprotein (HDL), cells were washed, kept in serum-free medium overnight and subsequently incubated with 10–100 mg/ml n-LDL, ox-LDL and HDL in fresh serum-free DMEM for 24 h. Controls were incubated under identical conditions without n-LDL, ox-LDL and HDL supplementation of the medium. 2.4. Senescence-associated beta-galactosidase activity RPE cells were subjected to acid b-galactosidase staining as described by Dimri and coworkers (Dimri et al., 1995). Briefly, treated RPE cells were washed twice with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS at pH 6.0 at room temperature (RT) for 4 min. Cells were then washed twice with PBS and incubated under lightprotection for 8 h at 37 C with fresh senescence-associated b-galactosidase staining solution (1 mg/ml 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside (X-gal), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl2, 2 mM MgCl2 diluted in PBS). Cells were then examined for the development of blue colour and photographed at low magnification (200) using a light microscope. All experiments were performed at least in triplicate in RPE cultures from three donors. 2.5. RNA isolation and real-time PCR Total RNA was isolated from 10 cm Petri dishes by the guanidium thiocyanate–phenol–chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the RNA samples was confirmed by electrophoresis in 1% Tris–acetate–EDTA (TAE)–agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed into cDNA by
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
reverse transcriptase. This cDNA was then used for quantitative real-time PCR. Quantification of human fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2), and transforming growth factor-beta2 (TGF-b2) mRNA was performed with specific primers (Table 1) on a LightCycler Instrument (LightCycler System, Roche Diagnostics, Germany). Primers and probes were selected with the programme ProbeFinder Version: 2.04. The cDNA of cells either treated with 10–50 mg/ml n-LDL, ox-LDL or HDL for 24 h was amplified with specific primers during 40 cycles. The standard curve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, 18S rRNA was simultaneously quantified in the same sample as an internal control. The level of Fn, Laa1, Col4a2, and TGF-b2 mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Fn, Laa1, Col4a2, and TGF-b2 mRNA by the level of the 18S rRNA housekeeping gene in the same samples. All experiments were performed at least in triplicate in RPE cultures from three donors. 2.6. Cell viability assay Cell viability was quantified based on a two-colour fluorescence assay, in which the nuclei of nonviable cells appear red due to staining by the membrane-impermeable dye propidium iodide (Sigma), whereas the nuclei of all cells were stained with the membrane-permeable dye Hoechst 33342 (Intergen, Purchase, NY). Confluent cultures of RPE cells growing on coverslips in 4 well tissue culture plates were exposed to 10–100 mg/ml n-LDL, ox-LDL and HDL for 24 h. For evaluation of cell viability, cells were washed in PBS and incubated with 2.0 mg/ml propidium iodide and 1.0 mg/ ml Hoechst 33342 for 20 min at 37 C. Subsequently, cells were analyzed under a fluorescence microscope (Leica DMR, Leica Microsystems, Wetzlar, Germany). Representative areas were documented with Leica IM 1000 software (Leica Microsystems, Heerbrugg, Switzerland). The labelled nuclei were then counted in fluorescence photomicrographs, and dead cells were expressed as a percentage of total nuclei in the field. The data are based on counts from nine experiments of RPE cultures of three donors performed in duplicate wells, with 3–5 documented representative fields per well. 2.7. Analysis of TGF-b2 in cell supernatants Levels of total TGF-b2 in supernatants were determined by a solid-phase TGF-b2 specific sandwich enzyme-linked immunosorbent assay (ELISA) (Quantikine, R&D System, Minneapolis, MN, USA). Briefly, cell supernatants were activated with 1.0 N HCl and subsequently neutralized with 1.2 N NaOH/0.5 M HEPES and analyzed using ELISA. All experiments were performed at least in triplicate in RPE cultures from three donors. The intra-assay and inter-assay coefficients of variation determined in our laboratory were 9.8% and 16%, respectively.
497
(10 mg/ml ox-LDL: 32.3 18.1%; 20 mg/ml ox-LDL: 32.9 17.9%; and 50 mg/ml ox-LDL: 50.2 19.7% of total cells) (Fig. 1C,E). In contrast, no effects on advanced senescence of RPE cells were observed after incubation with 10, 20 and 50 mg/ml HDL (10 mg/ml HDL: 2.5 1.1%; 20 mg/ml HDL: 2.5 1.0%; 50 mg/ml HDL: 2.0 0.9%) (Fig. 1D,E). 3.2. Ox-LDL and n-LDL induce the expression of ECM components We used real-time PCR to detect mRNA expression of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) after treatment with 10, 20 and 50 mg/ml of n-LDL, ox-LDL and HDL. The signals generated in untreated control cells were set to 1.0 by real-time PCR analysis (Fig. 2). The most prominent effect of n-LDL and ox-LDL was observed at a concentration of 50 mg/ml. Exposure of RPE cells to 50 mg/ml n-LDL for 24 h upregulated the mRNA expression of Fn by 2.5 0.3 fold (Fig. 2A), Laa1 by 3.2 0.9 fold (Fig. 2B), and Col4a2 by 2.5 0.3 fold (Fig. 2C). Treatment with 50 mg/ml ox-LDL increased Fn mRNA 3.2 0.6 fold (Fig. 2A), Laa1 mRNA 3.4 0.6 fold (Fig. 2B), and Col4a2 mRNA expression 2.3 0.6 fold (Fig. 2C). Concentrations of 10 and 20 mg/ml n-LDL and ox-LDL also led to elevated mRNA levels of the three ECM genes investigated (10 mg/ml n-LDL: Fn by 1.5 0.4 fold, Laa1 by 2.8 0.6 fold, Col4a2 by 1.8 0.4 fold; 20 mg/ml n-LDL: Fn by 1.8 0.4 fold, Laa1 by 2.9 0.7 fold, Col4a2 by 2.2 0.4 fold; 10 mg/ml ox-LDL: Fn by 2.7 0.5 fold, Laa1 by 2.9 0.7 fold, Col4a2 by 1.9 0.5 fold; 20 mg/ml ox-LDL: Fn by 2.4 0.6 fold, Laa1 by 2.8 0.8 fold, Col4a2 by 1.7 0.6 fold) (Fig. 2A,B,C). 3.3. Ox-LDL induces cell death in RPE cells To determine cytotoxic concentrations of n-LDL, ox-LDL and HDL, cultured human RPE cells were first exposed to various doses of n-LDL, ox-LDL and HDL, and then nonviable cells were detected by live dead assay. In untreated control cells, almost no dead cells staining red by propidium iodide were detectable (Fig. 3A,J). Incubation of cultured RPE cells with 10, 20 and 50 mg/ml n-LDL had minimal effects on RPE cell death (10 mg/ml n-LDL: 2.1 0.5%; 20 mg/ml n-LDL: 2.3 0.8%; 50 mg/ml n-LDL: 4.1 1%), whereas treatment with 100 mg/ml n-LDL increased the number of dead cells to 12.1 2.1% (Fig. 3B,J). Treatment with 10, 20 and 50 mg/ml ox-LDL for 24 h showed elevated proportions of nonviable cells in a dosedependent fashion (10 mg/ml ox-LDL: 3.2 1.5%; 20 mg/ml ox-LDL: 4.2 2%; 50 mg/ml ox-LDL: 12.3 4%) (Fig. 3J). Exposure to 100 mg/ ml ox-LDL markedly increased the proportion of nonviable RPE cells to 87.0 6% of total RPE cells (Fig. 3C,J). Treatment of RPE cells with HDL did not show any marked cytotoxic effects (10 mg/ml HDL: 2.0 0.6%; 20 mg/ml HDL: 2.2 0.7%; 50 mg/ml HDL: 2.1 0.9%; 100 mg/ml HDL: 3.0 0.8%) (Fig. 3D,J). Therefore, the experiments on further effects of all three lipoproteins in RPE cells were conducted with concentrations of 10, 20 and 50 mg/ml of n-LDL, ox-LDL and HDL. 3.4. Ox-LDL and n-LDL increase TGF-b2 expression in RPE cells
3. Results 3.1. Ox-LDL induces advanced senescence in RPE cells The effects of lipoproteins on advanced senescence in cultured RPE cells was evaluated by using b-galactosidase staining. In untreated controls, only single RPE cells expressed this enzyme (2.9 2.3% of total cells) (Fig. 1A,E). Treatment with 10, 20 and 50 mg/ml n-LDL for 24 h had minimal effects on b-galactosidase activity compared to untreated control cells (10 mg/ml n-LDL: 5.8 2.7%; 20 mg/ml n-LDL: 7.0 3.6%; 50 mg/ml n-LDL: 9.8 5.3%) (Fig. 1B,E). However, incubation with 10, 20 and 50 mg/ml ox-LDL for 24 h upregulated the number of b-galactosidase positive cells
In a subsequent approach, we investigated the effect of n-LDL, ox-LDL and HDL on the mRNA expression of transforming growth factor-beta2 (TGF-b2). The signals generated by real-time PCR analysis of untreated control cells were set to 1.0 (Fig. 4). Exposure to 10 and 20 mg/ml of n-LDL had no effect on the TGF-b2 mRNA expression (10 mg/ml n-LDL: 1.0 0.02 fold; 20 mg/ml n-LDL: 1.0 0.2 fold), whereas 50 mg/ml of n-LDL increased TGF-b2 mRNA amount by 1.7 0.3 fold compared to untreated control cells (Fig. 4A). Treatment of RPE cells with 10, 20 and 50 mg/ml of ox-LDL for 24 h under serum-free medium conditions increased the mRNA expression of TGF-b2 dose-dependently (10 mg/ml ox-LDL: 1.7 0.2 fold; 20 mg/ml ox-LDL: 2.4 0.4 fold; and 50 mg/ml ox-LDL:
498
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
4. Discussion
Table 1 Primers used for real-time PCR. Gene target
Gene sequence
Gene position
Fibronectin
50 -ctggccgaaaatacattgtaaa-30 50 -ccacagtcgggtcaggag-30
2611–2632 2707–2724
Laminin alpha 1
50 -ctaagctggctcccgatg-30 50 -catccaccacacactgttctg-30
8130–8147 8187–8207
Collagen type IV alpha 2
50 -gggatatttggcctgaaagg-30 50 -caggaagagcagcagaacct-30
4147–4166 4191–4210
TGF-b2
50 -caaagggtacaatgccaactt-30 50 -cagatgcttctggatttatggtatt-30
1195–1215 1283–1307
18S rRNA
50 -ctcaacacgggaaacctcac-30 50 -cgctccaccaactaagaacg-30
1348–1367 1438–1457
4.2 0.4 fold) (Fig. 4A). Treatment of RPE cells with up to 50 mg/ml of HDL did not induce any changes of TGF-b2 mRNA expression compared to untreated control cells (10 mg/ml n-LDL: 1.0 0.1 fold; 20 mg/ml n-LDL: 0.9 0.1 fold; 50 mg/ml n-LDL: 0.8 0.2 fold) (Fig. 4A). To determine total TGF-b2 protein levels released into culture medium after treatment with lipoproteins, a commercially available enzyme-linked immunosorbent assay (ELISA) was used. We found a basal level of 45 8 pg/ml TGF-b2 protein in untreated control cells (Fig. 4B). Treatment of RPE cells with 50 mg/ml n-LDL and 50 mg/ ml ox-LDL increased the TGF-b2 protein secretion to 196 35 pg/ml and 396 32 pg/ml (Fig. 4B). Lower concentrations of n-LDL and oxLDL also increased TGF-b2 protein expression (10 mg/ml n-LDL: 78 5 pg/ml; 20 mg/ml n-LDL: 120 23 pg/ml.; 10 mg/ml ox-LDL: 120 10 pg/ml, 20 mg/ml ox-LDL: 240 38 pg/ml) (Fig. 4B). We could not detect any increase of TGF-b2 protein levels after exposure to HDL (10 mg/ml HDL: 48 6 pg/ml; 20 mg/ml HDL: 44 14 pg/ml; 50 mg/ml HDL: 47 18 pg/ml) (Fig. 4B).
To examine the possible involvement of native low-density lipoprotein (n-LDL), oxidized low-density lipoprotein (ox-LDL) and high-density lipoprotein (HDL) in the pathogenesis of age-related macular degeneration (AMD), we have chosen to study their influence on four representative characteristics of different stages of AMD. In the present study, we demonstrated that n-LDL and more potently ox-LDL induce senescent changes, synthesis of extracellular matrix (ECM) components, cell loss, and expression of transforming growth factor-beta2 (TGF-b2) in the retinal pigment epithelium (RPE) in vitro. Until now, the sequence of cellular events leading to the progression of AMD is still under debate. One theoretical concept implies that senescent changes in the outer retina, the RPE, the Bruch’s membrane and the choroids might be the underlying process for the development of AMD (Roth et al., 2004; Zarbin, 2004). For the pathogenesis of both AMD and arteriosclerosis, it is assumed that increased senescent changes actually progress to the stage of a degenerative disease (Minamino and Komuro, 2007; Young, 1987). Previously, it has been demonstrated that advanced senescence is inducible by oxidative stress in cultured human RPE cells (Honda et al., 2002; Nilsson et al., 2003). In our experiments, ox-LDL promoted advanced senescence of cultured human RPE cells. In contrast, n-LDL and HDL had no effect on advanced senescence. In cultured human endothelial cells, advanced senescence has also been induced by ox-LDL (Imanishi et al., 2004; Maier et al., 1996). This observation is in accordance with endothelial senescence as seen in arteriosclerosis (Minamino and Komuro, 2007; Shi et al., 2007). Whether or not senescent changes in the RPE, as described for AMD (Feher et al., 2006), are induced by ox-LDL is still unknown. Advanced senescence of RPE cells eventually leads to RPE dysfunction, which is majorly manifested by incomplete degradation and increased aggregation of extracellular matrix (ECM)
Fig. 1. RPE cell senescence was evaluated by SA-b-Gal activity after treatment with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Representative photomicrographs show SA-b-Gal positive cells staining blue in RPE cells either (A) untreated or treated with 50 mg/ml (B) n-LDL, (C) ox-LDL or (D) HDL. (E) Quantification of the SA-b-Gal activity assay. The percentage of SA-b-Gal positive cells was scored by counting at least 700 cells in phase-contrast photomicrographs of representative fields. Data (mean s.d.) are based on the sampling of 6–10 photomicrographs per condition in three independent experiments with three different cell cultures from different donors (*P < 0.05). Scale bar: 100 mm.
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
499
Fig. 2. Analysis of lipoprotein effects on mRNA expression of (A) fibronectin (Fn), (B) laminin alpha 1 (Laa1) and (C) collagen type IV alpha 2 (Col4a2) in cultured human RPE cells by real-time PCR. Cells were treated with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Results were normalized to 18S rRNA as reference. Results are given as mean s.d. of three independent experiments with three different cell cultures from different donors (*P < 0.05).
deposits (Young, 1987; Zarbin, 2004). ECM formation belongs to the key pathological factors of AMD and is also known to be inducible by oxidative stress (Roth et al., 2004; Zarbin, 2004). We have demonstrated that both n-LDL and ox-LDL lead to induced expression of ECM components such as fibronectin (Fn), laminin alpha 1 (Laa1) and collagen type IV alpha 2 (Col4a2) in cultured human RPE cells. Previous studies showed that n-LDL and ox-LDL can act profibrogenic by stimulating ECM synthesis in mesangial
cells (Lee, 1999; Roh et al., 1998), human coronary artery smooth muscle cells (Bachem et al., 1999), human glomerular epithelial (Ding et al., 1997) and human hepatic stellate cells (Schneiderhan et al., 2001). The causal relationship between ox-LDL and ECM alterations is supported by their close spatial relation as found in the thickened intima of human coronary arteries (Fukuchi et al., 2002). Whether or not internalized ox-LDL in RPE cells triggers ECM synthesis in vivo is still unclear.
500
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
Fig. 3. Live dead assay of cultured human RPE cells treated with 10–100 mg/ml lipoproteins. Representative fluorescence photomicrographs of nonviable RPE cells show either (A) untreated or treated with 100 mg/ml (B) native (n)-LDL, (C) oxidized (ox)-LDL or (D) HDL. (E–H) Total number of RPE cells in the corresponding fields. (J) Quantification of the effects of lipoprotein treatment on the number of nonviable cells. The percentage of dead cells was scored by counting at least 700 cells in fluorescence photomicrographs of representative fields. Data (mean s.d.) are based on the sampling of 6–10 photomicrographs per condition in three independent experiments with three different cell cultures from different donors (*P < 0.05). Scale bar: 100 mm.
In face of the previous described pathological events such as cellular senescence and ECM formation, deteriorating RPE function may ultimately progress to RPE degeneration with RPE cell death (Young, 1987; Zarbin, 2004). RPE cell death is the manifestation of the most prevalent atrophic form of AMD (Petrukhin, 2007). In our experiments, ox-LDL markedly induced cell death in cultured human RPE cells. A lower cytotoxic effect was observed for n-LDL. Similarly, higher cytotoxic potency of ox-LDL compared to n-LDL has been described in other cellular systems such as human coronary artery smooth muscle cells (Bachem et al., 1999), coronary arterial endothelial cells (Li et al., 1998), and monocytes–macrophages (Hardwick et al., 1996). A number of studies have already demonstrated the cytotoxicity of ox-LDL in cultured human RPE cells with a similar treatment regimen (Gordiyenko et al., 2004; Hoppe et al., 2004; Rodriguez et al., 2004). In the study of Rodriguez et al. (2004), it was assumed that the formation of 7-ketocholesterol, an oxidized lipid in ox-LDL particles, may be responsible for the cytotoxicity of ox-LDL (Rodriguez et al., 2004). Further extended investigations are required to single out the specific component of ox-LDL underlying the cytotoxicity of ox-LDL in human RPE cells. It is still controversial whether and how pathogenic events such as senescent RPE changes, ECM deposition and RPE dysfunction elicit RPE atrophy (‘dry’ AMD) or choroidal neovascularization (‘wet’ AMD) or rather provoke both forms simultaneously (Young, 1987; Zarbin, 2004). One common finding of ‘dry’ and ‘wet’ AMD is the increased expression of TGF-b observed in the RPE cells both of human maculae with ‘dry’ AMD and of surgically removed neovascular membranes from eyes of ‘wet’ AMD (Kliffen et al., 1997). In
‘dry’ AMD, TGF-b has been found to be a promoting factor for ECM formation (Kliffen et al., 1997). Therefore, we have examined the influence of LDL and ox-LDL on TGF-b expression. In our study, both ox-LDL and, in a less marked manner, n-LDL increased TGF-b2 expression in cultured human RPE cells. Similarly, in cultured human endothelial (Maier et al., 1996) or glomerular epithelial cells (Ding et al., 1997), ox-LDL is able to induce increased levels of TGFb2. In contrast, treatment with high-density lipoprotein (HDL) had no effects on TGF-b2 levels in our experiments. To our knowledge, no data are yet available demonstrating the influence of lipoproteins on TGF-b2 production in the cultured human RPE cells. This information could help us to better understand the role of TGF-b2 in the human RPE in vivo. The source and mechanism of deposition of LDL and ox-LDL in the RPE cells are still obscure. On the one hand, there is evidence that de novo synthesis of lipoproteins by the RPE may occur (Fliesler et al., 1993; Malek et al., 2003). On the other hand, an external pathway for plasma lipoprotein accumulation via the fenestrated choroidal cell junctions and Bruch’s membrane into the RPE cells has been suggested in the in vivo situation (Gordiyenko et al., 2004). Concerning cultured human RPE cells, ox-LDL is internalized by CD36 scavenger receptors. With immunohistochemical and real-time PCR analysis we were able to demonstrate the expression of the CD36 scavenger receptor in our primary RPE cells (data not shown). These results are in accordance with a number of studies demonstrating that CD36 scavenger receptors are expressed both in the RPE (Gordiyenko et al., 2004; Ryeom et al., 1996; Yesner et al., 1996) and vascular endothelial cells (Boullier et al., 2001). In the pathogenesis of
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
501
Fig. 4. (A) Analysis of lipoprotein effects on TGF-b2 mRNA expression in cultured human RPE cells by real-time PCR. Cells were treated with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Results were normalized to 18S rRNA as reference. Results are given as mean s.d. of three independent experiments with three different cell cultures from different donors (*P < 0.05). (B) TGF-b2 protein secretion by RPE cells treated as in (A) and analyzed by ELISA assay. Results expressed as described in (A).
arteriosclerosis, the expression of CD36 scavenger receptors is the major route of internalization of ox-LDL (Boullier et al., 2001). Other well-known ligands for CD36 scavenger receptors are modified lipids of rod outer segment (ROS) membranes (Ryeom et al., 1996). ROS are shed daily from the distal ends of the photoreceptors by RPE cells (Sarangarajan and Apte, 2005). The process of degradation of internalized ROS involves the process of lysosome– phagosome fusion (Sarangarajan and Apte, 2005). CD36 scavenger receptors participate in the recognition and internalization of ROS (Ryeom et al., 1996). ROS membranes, which are rich in long chain polyunsaturated fatty acids, can be modified by lipid peroxidation leading to the creation of oxidized ROS (Hoppe et al., 2004; Ryeom et al., 1996). Since both ox-ROS and ox-LDL are recognized by CD36 scavenger receptors and may perturb the lysosomal function of the RPE (Hoppe et al., 2004; Sarangarajan and Apte, 2005), it is tempting to speculate that the possible involvement of ox-LDL in the induction of AMD-like changes in this in vitro model might actually represent changes that are in vivo caused by ox-ROS. In conclusion, our findings showed that n-LDL and, in a greater extent, ox-LDL induce senescent changes, synthesis of ECM components, cell loss, and expression of TGF-b2 in cultured human RPE cells. In the in vivo situation, these pathogenic cellular events are shared by AMD and atherosclerosis. Our experiments demonstrated that n-LDL and more significantly ox-LDL induce cellular events in cultured human RPE cells, which may resemble pathogenic changes in AMD. Therefore, this study may provide some
fundamental results needed for further investigations on the effects of lipoproteins in the RPE. Acknowledgements The authors thank B. Zimmer, J. Moriniere and K. Obholzer for excellent technical assistance, Harald Kroehn for preparing the figures. This work was supported by DFG WE 2577/2-1 and DOG Research Support to UW-L. References Alge, C.S., Priglinger, S.G., Neubauer, A.S., Kampik, A., Zillig, M., Bloemendal, H., Welge-Lussen, U., 2002. Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin. Invest. Ophthalmol. Vis. Sci. 43, 3575–3582. Al-Shaer, M.H., Choueiri, N.E., Correia, M.L., Sinkey, C.A., Barenz, T.A., Haynes, W.G., 2006. Effects of aging and atherosclerosis on endothelial and vascular smooth muscle function in humans. Int. J. Cardiol. 109, 201–206. An, E., Lu, X., Flippin, J., Devaney, J.M., Halligan, B., Hoffman, E.P., Strunnikova, N., Csaky, K., Hathout, Y., 2006. Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors. J. Proteome Res. 5, 2599–2610. Bachem, M.G., Wendelin, D., Schneiderhan, W., Haug, C., Zorn, U., Gross, H.J., SchmidKotsas, A., Gru¨nert, A., 1999. Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells. Clin. Chem. Lab. Med. 37, 319–326. Boullier, A., Bird, D.A., Chang, M.K., Dennis, E.A., Friedman, P., Gillotre-Taylor, K., Ho¨rkko¨, S., Palinski, W., Quehenberger, O., Shaw, P., Steinberg, D., Terpstra, V.,
502
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
Witztum, J.L., 2001. Scavenger receptors, oxidized LDL and atherosclerosis. Ann. N. Y. Acad. Sci. 947, 214–222. Campochiaro, P.A., Jerdon, J.A., Glaser, B.M., 1986. The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro. Invest. Ophthalmol. Vis. Sci. 27, 1615–1621. Chamberlain, M., Baird, P., Dirani, M., Guymer, R., 2006. Unraveling a complex genetic disease: age-related macular degeneration. Surv. Ophthalmol. 51, 576–586. Cipollone, F., Fazia, M., Mincione, G., Iezzi, A., Pini, B., Cuccurullo, C., Ucchino, S., Spigonardo, F., Di Nisio, M., Cuccurullo, F., Mezzetti, A., Porreca, E., 2004. Increased expression of transforming growth factor-beta1 as a stabilizing factor in human atherosclerotic plaques. Stroke 35, 2253–2257. DeWan, A., Bracken, M.B., Hoh, J., 2007. Two genetic pathways for age-related macular degeneration. Curr. Opin. Genet. Dev. 17, 228–233. Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U. S. A. 92, 9363–9367. Ding, G., van Goor, H., Ricardo, S.D., Orlowski, J.M., Diamond, J.R., 1997. Oxidized LDL stimulates the expression of TGF-beta and fibronectin in human glomerular epithelial cells. Kidney Int. 51, 147–154. Edwards, A.O., Malek, G., 2007. Molecular genetics of AMD and current animal models. Angiogenesis 10, 119–132. Feher, J., Kovacs, I., Artico, M., Cavallotti, C., Papale, A., Balacco Gabrieli, C., 2006. Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration. Neurobiol. Aging 27, 983–993. Fliesler, S.J., Florman, R., Rapp, L.M., Pittler, S.J., Keller, R.K., 1993. In vivo biosynthesis of cholesterol in the rat retina. FEBS Lett. 335, 234–238. Fukuchi, M., Watanabe, J., Kumagai, K., Baba, S., Shinozaki, T., Miura, M., Kagaya, Y., Shirato, K., 2002. Normal and oxidized low density lipoproteins accumulate deep in physiologically thickened intima of human coronary arteries. Lab. Invest. 82, 1437–1447. Godley, B.F., Jin, G.F., Guo, Y.S., Hurst, J.S., 2002. Bcl-2 overexpression increases survival in human retinal pigment epithelial cells exposed to H2O2. Exp. Eye Res. 74, 663–669. Gordiyenko, N., Campos, M., Lee, J.W., Fariss, R.N., Sztein, J., Rodriguez, I.R., 2004. RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 45, 2822–2829. Guymer, R.H., Chiu, A.W., Lim, L., Baird, P.N., 2005. HMG CoA reductase inhibitors (statins): do they have a role in age-related macular degeneration? Surv. Ophthalmol. 50, 194–206. Hardwick, S.J., Hegyi, L., Clare, K., Law, N.S., Carpenter, K.L., Mitchinson, M.J., Skepper, J.N., 1996. Apoptosis in human monocyte–macrophages exposed to oxidized low density lipoprotein. J. Pathol. 179, 294–302. Hawkins, B.S., Bird, A., Klein, R., West, S.K., 1999. Epidemiology of age-related macular degeneration. Mol. Vis. 5, 26. Holz, F.G., Sheraidah, G., Pauleikhoff, D., Bird, A.C., 1994. Analysis of lipid deposits extracted from human macular and peripheral Bruch’s membrane. Arch. Ophthalmol. 112, 402–406. Honda, S., Hjelmeland, L.M., Handa, J.T., 2002. Senescence associated beta galactosidase activity in human retinal pigment epithelial cells exposed to mild hyperoxia in vitro. Br. J. Ophthalmol. 86, 159–162. Hoppe, G., O’Neil, J., Hoff, H.F., Sears, J., 2004. Accumulation of oxidized lipid– protein complexes alters phagosome maturation in retinal pigment epithelium. Cell Mol. Life Sci. 61, 1664–1674. Hyman, L., Neborsky, R., 2002. Risk factors for age-related macular degeneration: an update. Curr. Opin. Ophthalmol. 13, 171–175. Imanishi, T., Hano, T., Sawamura, T., Nishio, I., 2004. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. Clin. Exp. Pharmacol. Physiol. 31, 407–413. Kamei, M., Yoneda, K., Kume, N., Suzuki, M., Itabe, H., Matsuda, K., Shimaoka, T., Minami, M., Yonehara, S., Kita, T., Kinoshita, S., 2007. Scavenger receptors for oxidized lipoprotein in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 48, 1801–1807. Klein, R., Klein, B.E., Linton, K.L.P., 1992. Prevalence of age-related maculopathy: the Beaver Dam Eye Study. Ophthalmology 99, 933–943. Klein, R., Klein, B.E., Tomany, S.C., Cruickshanks, K.J., 2003. The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam Eye study. Ophthalmology 110, 636–643. Klein, R., Peto, T., Bird, A., Vannewkirk, M.R., 2004. The epidemiology of age-related macular degeneration. Am. J. Ophthalmol. 137, 486–495. Kliffen, M., Sharma, H.S., Mooy, C.M., Kerkvliet, S., de Jong, P.T., 1997. Increased expression of angiogenic growth factors in age-related maculopathy. Br. J. Ophthalmol. 81, 154–162. Lee, H.S., 1999. Oxidized LDL, glomerular mesangial cells and collagen. Diabetes Res. Clin. Pract. 45, 117–122. van Leeuwen, R., Klaver, C.C., Vingerling, J.R., Hofman, A., de Jong, P.T., 2003. Epidemiology of age-related maculopathy: a review. Eur. J. Epidemiol. 18, 845–854. Leschey, K.H., Hackett, S.F., Singer, J.H., Campochiaro, P.A., 1990. Growth factor responsiveness of human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 31, 839–846. Li, D., Yang, B., Mehta, J.L., 1998. Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas. Am. J. Physiol. 275, 568–576. Maier, J.A., Barenghi, L., Bradamante, S., Pagani, F., 1996. Induction of human endothelial cell growth by mildly oxidized low density lipoprotein. Atherosclerosis 123, 115–121.
Malek, G., Li, C.M., Guidry, C., Medeiros, N.E., Curcio, C.A., 2003. Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with agerelated maculopathy. Am. J. Pathol. 162, 413–425. Mares-Perlman, J.A., Brady, W.E., Klein, R., VandenLangenberg, G.M., Klein, B.E., Palta, M., 1995. Dietary fat and age-related maculopathy. Arch. Ophthalmol. 113, 743–748. Minamino, T., Miyauchi, H., Yoshida, T., Tateno, K., Komuro, I., 2004. The role of vascular cell senescence in atherosclerosis: antisenescence as a novel therapeutic strategy for vascular aging. Curr. Vasc. Pharmacol. 2, 141–148. Minamino, T., Komuro, I., 2007. Vascular cell senescence: contribution to atherosclerosis. Circ. Res. 100, 15–26. Mitchell, P., Wang, J.J., Smith, W., Leeder, S.R., 2002. Smoking and the 5-year incidence of age-related maculopathy: the Blue Mountains Eye study. Arch. Ophthalmol. 120, 1357–1363. Nakajima, K., Nakano, T., Tanaka, A., 2006. The oxidative modification hypothesis of atherosclerosis: the comparison of atherogenic effects on oxidized LDL and remnant lipoproteins in plasma. Clin. Chim. Acta 367, 36–47. Newby, A.C., 2005. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol. Rev. 85, 1–31. Nilsson, S.E., Sundelin, S.P., Wihlmark, U., Brunk, U.T., 2003. Aging of cultured retinal pigment epithelial cells: oxidative reactions, lipofuscin formation and blue light damage. Doc. Ophthalmol. 106, 13–16. Petrukhin, K., 2007. New therapeutic targets in atrophic age-related macular degeneration. Expert Opin. Ther. Targets 11, 625–639. Rahaman, S.O., Lennon, D.J., Febbraio, M., Podrez, E.A., Hazen, S.L., Silverstein, R.L., 2006. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell Metab. 4, 211–221. Robman, L., McNeil, J., Dimitrov, P., Dowrick, A., Tikellis, G., Nicolas, C., Cameron, J., Guymer, R., McGrath, B., McCarty, C., 2004. Methodology of the Cardiovascular Health and Age-Related Maculopathy (CHARM) Study. Ophthalmic Epidemiol. 11, 161–179. Rodriguez, I.R., Alam, S., Lee, J.W., 2004. Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol. Invest. Ophthalmol. Vis. Sci. 45, 2830–2837. Roh, D.D., Kamanna, V.S., Kirschenbaum, M.A., 1998. Oxidative modification of lowdensity lipoprotein enhances mesangial cell protein synthesis and gene expression of extracellular matrix proteins. Am. J. Nephrol. 18, 344–350. Roth, F., Bindewald, A., Holz, F.G., 2004. Keypathophysiologic pathways in agerelated macular disease. Graefe’s Arch. Clin. Exp. Ophthalmol. 242, 710–716. Ruberti, J.W., Curcio, C.A., Millican, C.L., Bailey, T., Kruth, H.S., 2001. Accumulation of cholesterol with age in human Bruch’s membrane. Invest. Ophthalmol. Vis. Sci. 42, 265–274. Rubic, T., Lorenz, R.L., 2006. Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10. Cardiovasc. Res. 69, 527–535. Ryeom, S.W., Silverstein, R.L., Scotto, A., Sparrow, J.R., 1996. Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36. J. Biol. Chem. 271, 20536–20539. Sarangarajan, R., Apte, S.P., 2005. Review melanization and phagocytosis: implications for age related macular degeneration. Mol. Vis. 11, 482–490. van der Schaft, T.L., Mooy, C.M., de Bruijn, W.C., Bosman, F.T., de Jong, P.T., 1994. Immunohistochemical light and electron microscopy of basal laminar deposit. Graefe’s Arch. Clin. Exp. Ophthalmol. 232, 40–46. Schmidt, S., Qin, X., Schmidt, M.A., Martin, E.R., Hauser, E.R., 2007. Interpreting analyses of continuous covariates in affected sibling pair linkage studies. Genet. Epidemiol. 31, 541–552. Schneiderhan, W., Schmid-Kotsas, A., Zhao, J., Gru¨nert, A., Nu¨ssler, A., Weidenbach, H., Menke, A., Schmid, R.M., Adler, G., Bachem, M.G., 2001. Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis. Hepatology 34, 729–737. Schulz, T., Schiffl, H., Scheithe, R., Hrboticky, N., Lorenz, R., 1995. Preserved antioxidative defense of lipoproteins in renal failure and during hemodialysis. Am. J. Kidney Dis. 25, 564–571. Schwartz, C.J., Valente, A.J., Sprague, E.A., Kelley, J.L., Nerem, R.M., 1991. The pathogenesis of atherosclerosis: an overview. Clin. Cardiol. 14, 11–16. Shi, Q., Hubbard, G.B., Kushwaha, R.S., Rainwater, D., Thomas 3rd, C.A., Leland, M.M., Vandeberg, J.L., Wang, X.L., 2007. Endothelial senescence after high-cholesterol, high-fat diet challenge in baboons. Am. J. Physiol. Heart Circ. Physiol. 292, 2913–2920. Sivaprasad, S., Bailey, T.A., Chong, V.N., 2005. Bruch’s membrane and the vascular intima: is there a common basis for age-related changes and disease? Clin. Exp. Ophthalmol. 33, 518–523. Smith, W., Assink, J., Klein, R., Mitchell, P., Klaver, C.C., Klein, B.E., Hofman, A., Jensen, S., Wang, J.J., de Jong, P.T., 2001. Risk factors for age-related macular degeneration: pooled findings from three continents. Ophthalmology 108, 697–704. Stoneman, V.E., Bennett, M.R., 2004. Role of apoptosis in atherosclerosis and its therapeutic implications. Clin. Sci. (Lond.) 107, 343–354. Takahashi, Y., Zhu, H., Yoshimoto, T., 2005. Essential roles of lipoxygenases in LDL oxidation and development of atherosclerosis. Antioxid. Redox Signal 7, 425–431. Tan, J.S., Mitchell, P., Smith, W., Wang, J.J., 2007. Cardiovascular risk factors and the long-term incidence of age-related macular degeneration: the Blue Mountains Eye Study. Ophthalmology 114, 1143–1150. Thornton, J., Edwards, R., Mitchell, P., Harrison, R.A., Buchan, I., Kelly, S.P., 2005. Smoking and age-related macular degeneration: a review of association. Eye 19, 935–944.
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503 Tsimikas, S., 2006. Oxidized low-density lipoprotein biomarkers in atherosclerosis. Curr. Atheroscler. Rep. 8, 55–61. Vingerling, J.R., Dielemans, I., Hofman, A., Grobbee, D.E., Hijmering, M., Kramer, C.F., de Jong, P.T., 1995a. The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology 102, 205–210. Vingerling, J.R., Dielemans, I., Bots, M.L., Hofman, A., Grobbee, D.E., de Jong, P.T.V.M., 1995b. Age-related macular degeneration is associated with atherosclerosis: the Rotterdam Study. Am. J. Epidemiol. 142, 404–409. Weber, C., Draude, G., Weber, K.S., Wubert, J., Lorenz, R.L., Weber, P.C., 1999. Downregulation by tumor necrosis factor-alpha of monocyte CCR2 expression and monocyte chemotactic protein-1-induced transendothelial migration is antagonized by oxidized low-density lipoprotein: a potential
503
mechanism of monocyte retention in atherosclerotic lesions. Atherosclerosis 145, 115–123. Yesner, L.M., Huh, H.Y., Pearce, S.F., Silverstein, R.L., 1996. Regulation of monocyte CD36 and thrombospondin-1 expression by soluble mediators. Arterioscler. Thromb. Vasc. Biol. 16, 1019–1025. Young, R.W., 1987. Pathophysiology of age-related macular degeneration. Surv. Ophthalmol. 31, 291–306. Zarbin, M.A., 2004. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122, 598–614. Zhu, C., Ying, D., Zhou, D., Mi, J., Zhang, W., Chang, Q., Li, L., 2005. Expression of TGFbeta1 in smooth muscle cells regulates endothelial progenitor cells migration and differentiation. J. Surg. Res. 125, 151–156.
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells Alice L. Yu a, Reinhard L. Lorenz b, Christos Haritoglou a, Anselm Kampik a, Ulrich Welge-Lussen a, c, * a
Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336 Munich, Germany Institute for Prophylaxis of Cardiovascular Diseases, Ludwig-Maximilians-University, Pettenkoferstrasse 9, 80336 Munich, Germany c Department of Ophthalmology, Friedrich-Alexander-University, Schwabachanlage 6, 91054 Erlangen, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2008 Accepted in revised form 30 October 2008 Available online 27 November 2008
Age-related macular degeneration (AMD) and artherosclerosis share common characteristics in their pathogenesis. In this study, we investigated the effects of lipoproteins like native (n)-LDL, oxidized (ox)LDL and high-density lipoprotein (HDL) on advanced senescence, extracellular matrix accumulation, cell loss, and transforming growth factor-beta2 (TGF-b2) expression in cultured human retinal pigment epithelial (RPE) cells. Primary human RPE cells were incubated with 10–100 mg/ml n-LDL, ox-LDL, and HDL for 24 h. For determination of advanced senescence, beta-galactosidase staining was used. The induction of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) mRNA was quantified by real-time PCR. Cell loss was investigated by live dead assay. Expression of TGF-b2 was analyzed by real-time PCR and ELISA assays. Ox-LDL accelerated dose-dependently the onset of RPE senescence, whereas LDL and HDL had no effect. LDL and ox-LDL led to induced expression of Fn, Laa1 and Col4a2, whereas HDL had no influence. Incubation of RPE cells with 100 mg/ml ox-LDL induced marked cell death compared to untreated control cells. Expression of TGF-b2 was dose-dependently increased by LDL and ox-LDL. LDL and ox-LDL induced cellular changes in RPE cells in vitro, which may resemble pathogenic events of AMD. These results may provide further information about the effects of LDL and ox-LDL in the human RPE and their potential role in the pathogenesis of AMD. Ó 2008 Published by Elsevier Ltd.
Keywords: lipoprotein retinal pigment epithelium age-related macular degeneration
1. Introduction Age-related macular degeneration (AMD) is the leading cause of visual loss in the elderly in Western societies (Hawkins et al., 1999; Klein et al., 1992; Vingerling et al., 1995a). Until now, the cause of AMD is still unclear. It is suspected that both genetic and environmental factors may play a role in the disease development and progression (Chamberlain et al., 2006; DeWan et al., 2007; Edwards and Malek, 2007; Schmidt et al., 2007). A number of epidemiological studies suggest that AMD shares common risk factors with atherosclerosis (Robman et al., 2004; Vingerling et al., 1995b) like smoking (Mitchell et al., 2002; Smith et al., 2001; Thornton et al., 2005; van Leeuwen et al., 2003), hypertension (Hyman and Neborsky, 2002; Klein et al., 2003, 2004) and elevated cholesterol (Guymer et al., 2005; Mares-Perlman et al., 1995; Tan et al., 2007).
* Corresponding author. Present address: Augenklinik der Friedrich-AlexanderUniversita¨t, Schwabachanlage 6, 91054 Erlangen, Germany. Tel.: þ49 9131 44731; fax: þ49 9131 8534630. E-mail address: [email protected] (U. Welge-Lussen). 0014-4835/$ – see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.exer.2008.10.028
The development of AMD is assumed to start from senescenceassociated RPE changes (Feher et al., 2006). AMD is further characterized by diffuse thickening of Bruch’s membrane due to extracellular matrix (ECM) accumulation such as fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) (An et al., 2006; van der Schaft et al., 1994). Bruch’s membrane represents a specialized vascular intima between the retinal pigment epithelium (RPE) and choriocapillaris (Sivaprasad et al., 2005). Further characteristic AMD changes include primary loss of RPE cells (Roth et al., 2004) and increased levels of transforming growth factor-beta (TGF-b) (Kliffen et al., 1997). The reason for these changes is still unknown. Pathological correlations have been found in atherosclerotic lesions in the intima of large arteries. In atherosclerotic plaque formation, similar events have been observed, such as advanced senescence of endothelial and smooth muscle cells (Al-Shaer et al., 2006; Minamino et al., 2004), diffuse intimal thickening (Newby, 2005), loss of vascular endothelial cells (Stoneman and Bennett, 2004), and increased expression of TGF-b (Cipollone et al., 2004; Zhu et al., 2005). Several lines of evidence suggest that in these atherosclerotic lesions, oxidative modification of low-density lipoprotein (LDL) in the vessel wall is the key event in atherogenesis
496
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
(Schwartz et al., 1991). This critical step is initiated by binding of LDL to the LDL receptor-related protein (LRP), a cell-surface receptor, and subsequent oxidation by 12/15-lipoxygenase (Takahashi et al., 2005). Oxidized low-density lipoprotein (ox-LDL) is the bioreactive variant of LDL and the major contributor for arteriosclerotic changes (Nakajima et al., 2006; Tsimikas, 2006). In atherogenesis, ox-LDL is internalized by activated macrophages via their scavenger receptors like CD36 leading to foam cell formation (Rahaman et al., 2006). In the eye, a broad range of evidence implicates that abnormal age-related accumulation of lipids and cholesterol takes place in the basal laminar deposits of the underlying Bruch’s membrane (Ruberti et al., 2001; Holz et al., 1994; Malek et al., 2003). It has been shown that the spatial distribution of accumulated lipids correlates with the lesions of AMD (Ruberti et al., 2001; Holz et al., 1994). This observation has suggested that lipids may be involved in the pathogenesis of AMD. Furthermore, the high metabolic activity of photoreceptor outer segments and the formation of free radicals are thought to implicate a high local turnover rate of oxidatively modified lipids in RPE (Godley et al., 2002). In previous studies, it has been demonstrated that CD36 scavenger receptors are also highly expressed in RPE cells (Gordiyenko et al., 2004; Ryeom et al., 1996; Yesner et al., 1996). Thus, there is growing evidence that LDL and ox-LDL may contribute to the pathogenesis of AMD (Gordiyenko et al., 2004; Kamei et al., 2007). However, until now, no studies exist to show whether or not LDL and ox-LDL have the potential to induce cellular changes as seen in AMD. The goal of the present study is to investigate the effects of native (n)-LDL and ox-LDL on characteristic RPE changes as found in AMD, such as senescent changes, synthesis of ECM components, cell loss, and expression of TGF-b2 and in cultured human RPE cells. 2. Methods 2.1. Isolation of human RPE cells Ten human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 4–16 h after death. The donors ranged in age from 38 to 63 years. None of the donors had a known history of eye disease. Methods of securing human tissue were humane, included proper consent and approval, complied with the declaration of Helsinki, and was approved by the local ethic committee. Human RPE cells were harvested following the procedure as described previously (Alge et al., 2002; Campochiaro et al., 1986). In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% polyvinylpyrrolidone iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in the sodium chloride solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera using fine forceps. The eyecup was rinsed with Ca2þ- and Mg2þ-free Hank’s balanced salt solution, and treated with 0.25% trypsin (GIBCO, Karlsruhe, Germany) for 1 h at 37 C. The trypsin solution was aspirated and replaced with Dulbecco´s modified eagles medium (DMEM, Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS) (Biochrom). Using a pipette, the media was gently agitated, releasing the RPE into the media by avoiding damage to Bruch’s membrane. 2.2. Lipoprotein preparation and oxidation Lipoproteins were prepared from 80 ml blood from healthy donors by gradient ultracentrifugation (Rubic and Lorenz, 2006; Schulz et al., 1995; Weber et al., 1999). In brief, plasma was carefully
pipetted into four ultracentrifugation tubes (polyallmore centrifuge tubes, Beckmann, Fullerton, CA) and overrun with a density gradient (3 ml d1 ¼1.080 g/ml and 3 ml d2 ¼ 1.050 g/ml of potassium bromide and ethylenediaminetetraacetic acid (EDTA) 0.1% (Titriplex III pa Merck, Germany) in aqua bidest and 2 ml d3 ¼ 1.0 g/ ml EDTA 0.1% in aqua bidest, pH 7.4). Gradient centrifugation was performed in a Beckmann L7-55 ultracentrifuge run at 36,000 rpm and 10 C for 24 h. LDL and HDL subfractions were aspirated from the gradient and stored at 4 C under nitrogen in darkness no longer than 8 h until oxidation. Before oxidation, native LDL was desalted using Econo Pac DG 10 chromatographic columns (Biorad, Munich, Germany). Then, native LDL was oxidized by incubation of EDTA-free LDL with 10 mM CuCl2 in phosphate-buffered saline (PBS) for 16 h at 37 C. The oxidation was monitored at 234 nm in an Uvikon 930 spectrophometer. After 16 h of incubation, 0.24 mM EDTA was added to stop oxidation. Protein content was quantified by a modification of Lowry’s method. Ox-LDL was concentrated with Centriflo Cones (BioRad) (2200 rpm, 20 min, 4 C) and washed twice with PBS buffer. 2.3. Human RPE cell culture The human RPE cell suspension was added to a 50 ml flask (Falcon, Wiesbaden, Germany) containing 20 ml of DMEM supplemented with 20% FCS and maintained at 37 C and 5% carbon dioxide (CO2). Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pan-cytokeratin antibody (Sigma, Deisenhofen, Germany) (Leschey et al., 1990). Cells were tested and found free of contaminating macrophages (anti-CD11, Sigma) and endothelial cells (anti-von Willbrand factor, Sigma). After reaching confluence, primary RPE cells were subcultured and maintained in DMEM supplemented with 10% FCS at 37 C and in CO2. Confluent primary RPE cells of passage 3–5 were used for the experiments. To test the effects of native low-density lipoprotein (n-LDL), oxidized low-density lipoprotein (ox-LDL), and highdensity lipoprotein (HDL), cells were washed, kept in serum-free medium overnight and subsequently incubated with 10–100 mg/ml n-LDL, ox-LDL and HDL in fresh serum-free DMEM for 24 h. Controls were incubated under identical conditions without n-LDL, ox-LDL and HDL supplementation of the medium. 2.4. Senescence-associated beta-galactosidase activity RPE cells were subjected to acid b-galactosidase staining as described by Dimri and coworkers (Dimri et al., 1995). Briefly, treated RPE cells were washed twice with PBS and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS at pH 6.0 at room temperature (RT) for 4 min. Cells were then washed twice with PBS and incubated under lightprotection for 8 h at 37 C with fresh senescence-associated b-galactosidase staining solution (1 mg/ml 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside (X-gal), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl2, 2 mM MgCl2 diluted in PBS). Cells were then examined for the development of blue colour and photographed at low magnification (200) using a light microscope. All experiments were performed at least in triplicate in RPE cultures from three donors. 2.5. RNA isolation and real-time PCR Total RNA was isolated from 10 cm Petri dishes by the guanidium thiocyanate–phenol–chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the RNA samples was confirmed by electrophoresis in 1% Tris–acetate–EDTA (TAE)–agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed into cDNA by
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
reverse transcriptase. This cDNA was then used for quantitative real-time PCR. Quantification of human fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2), and transforming growth factor-beta2 (TGF-b2) mRNA was performed with specific primers (Table 1) on a LightCycler Instrument (LightCycler System, Roche Diagnostics, Germany). Primers and probes were selected with the programme ProbeFinder Version: 2.04. The cDNA of cells either treated with 10–50 mg/ml n-LDL, ox-LDL or HDL for 24 h was amplified with specific primers during 40 cycles. The standard curve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, 18S rRNA was simultaneously quantified in the same sample as an internal control. The level of Fn, Laa1, Col4a2, and TGF-b2 mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Fn, Laa1, Col4a2, and TGF-b2 mRNA by the level of the 18S rRNA housekeeping gene in the same samples. All experiments were performed at least in triplicate in RPE cultures from three donors. 2.6. Cell viability assay Cell viability was quantified based on a two-colour fluorescence assay, in which the nuclei of nonviable cells appear red due to staining by the membrane-impermeable dye propidium iodide (Sigma), whereas the nuclei of all cells were stained with the membrane-permeable dye Hoechst 33342 (Intergen, Purchase, NY). Confluent cultures of RPE cells growing on coverslips in 4 well tissue culture plates were exposed to 10–100 mg/ml n-LDL, ox-LDL and HDL for 24 h. For evaluation of cell viability, cells were washed in PBS and incubated with 2.0 mg/ml propidium iodide and 1.0 mg/ ml Hoechst 33342 for 20 min at 37 C. Subsequently, cells were analyzed under a fluorescence microscope (Leica DMR, Leica Microsystems, Wetzlar, Germany). Representative areas were documented with Leica IM 1000 software (Leica Microsystems, Heerbrugg, Switzerland). The labelled nuclei were then counted in fluorescence photomicrographs, and dead cells were expressed as a percentage of total nuclei in the field. The data are based on counts from nine experiments of RPE cultures of three donors performed in duplicate wells, with 3–5 documented representative fields per well. 2.7. Analysis of TGF-b2 in cell supernatants Levels of total TGF-b2 in supernatants were determined by a solid-phase TGF-b2 specific sandwich enzyme-linked immunosorbent assay (ELISA) (Quantikine, R&D System, Minneapolis, MN, USA). Briefly, cell supernatants were activated with 1.0 N HCl and subsequently neutralized with 1.2 N NaOH/0.5 M HEPES and analyzed using ELISA. All experiments were performed at least in triplicate in RPE cultures from three donors. The intra-assay and inter-assay coefficients of variation determined in our laboratory were 9.8% and 16%, respectively.
497
(10 mg/ml ox-LDL: 32.3 18.1%; 20 mg/ml ox-LDL: 32.9 17.9%; and 50 mg/ml ox-LDL: 50.2 19.7% of total cells) (Fig. 1C,E). In contrast, no effects on advanced senescence of RPE cells were observed after incubation with 10, 20 and 50 mg/ml HDL (10 mg/ml HDL: 2.5 1.1%; 20 mg/ml HDL: 2.5 1.0%; 50 mg/ml HDL: 2.0 0.9%) (Fig. 1D,E). 3.2. Ox-LDL and n-LDL induce the expression of ECM components We used real-time PCR to detect mRNA expression of fibronectin (Fn), laminin alpha 1 (Laa1), and collagen type IV alpha 2 (Col4a2) after treatment with 10, 20 and 50 mg/ml of n-LDL, ox-LDL and HDL. The signals generated in untreated control cells were set to 1.0 by real-time PCR analysis (Fig. 2). The most prominent effect of n-LDL and ox-LDL was observed at a concentration of 50 mg/ml. Exposure of RPE cells to 50 mg/ml n-LDL for 24 h upregulated the mRNA expression of Fn by 2.5 0.3 fold (Fig. 2A), Laa1 by 3.2 0.9 fold (Fig. 2B), and Col4a2 by 2.5 0.3 fold (Fig. 2C). Treatment with 50 mg/ml ox-LDL increased Fn mRNA 3.2 0.6 fold (Fig. 2A), Laa1 mRNA 3.4 0.6 fold (Fig. 2B), and Col4a2 mRNA expression 2.3 0.6 fold (Fig. 2C). Concentrations of 10 and 20 mg/ml n-LDL and ox-LDL also led to elevated mRNA levels of the three ECM genes investigated (10 mg/ml n-LDL: Fn by 1.5 0.4 fold, Laa1 by 2.8 0.6 fold, Col4a2 by 1.8 0.4 fold; 20 mg/ml n-LDL: Fn by 1.8 0.4 fold, Laa1 by 2.9 0.7 fold, Col4a2 by 2.2 0.4 fold; 10 mg/ml ox-LDL: Fn by 2.7 0.5 fold, Laa1 by 2.9 0.7 fold, Col4a2 by 1.9 0.5 fold; 20 mg/ml ox-LDL: Fn by 2.4 0.6 fold, Laa1 by 2.8 0.8 fold, Col4a2 by 1.7 0.6 fold) (Fig. 2A,B,C). 3.3. Ox-LDL induces cell death in RPE cells To determine cytotoxic concentrations of n-LDL, ox-LDL and HDL, cultured human RPE cells were first exposed to various doses of n-LDL, ox-LDL and HDL, and then nonviable cells were detected by live dead assay. In untreated control cells, almost no dead cells staining red by propidium iodide were detectable (Fig. 3A,J). Incubation of cultured RPE cells with 10, 20 and 50 mg/ml n-LDL had minimal effects on RPE cell death (10 mg/ml n-LDL: 2.1 0.5%; 20 mg/ml n-LDL: 2.3 0.8%; 50 mg/ml n-LDL: 4.1 1%), whereas treatment with 100 mg/ml n-LDL increased the number of dead cells to 12.1 2.1% (Fig. 3B,J). Treatment with 10, 20 and 50 mg/ml ox-LDL for 24 h showed elevated proportions of nonviable cells in a dosedependent fashion (10 mg/ml ox-LDL: 3.2 1.5%; 20 mg/ml ox-LDL: 4.2 2%; 50 mg/ml ox-LDL: 12.3 4%) (Fig. 3J). Exposure to 100 mg/ ml ox-LDL markedly increased the proportion of nonviable RPE cells to 87.0 6% of total RPE cells (Fig. 3C,J). Treatment of RPE cells with HDL did not show any marked cytotoxic effects (10 mg/ml HDL: 2.0 0.6%; 20 mg/ml HDL: 2.2 0.7%; 50 mg/ml HDL: 2.1 0.9%; 100 mg/ml HDL: 3.0 0.8%) (Fig. 3D,J). Therefore, the experiments on further effects of all three lipoproteins in RPE cells were conducted with concentrations of 10, 20 and 50 mg/ml of n-LDL, ox-LDL and HDL. 3.4. Ox-LDL and n-LDL increase TGF-b2 expression in RPE cells
3. Results 3.1. Ox-LDL induces advanced senescence in RPE cells The effects of lipoproteins on advanced senescence in cultured RPE cells was evaluated by using b-galactosidase staining. In untreated controls, only single RPE cells expressed this enzyme (2.9 2.3% of total cells) (Fig. 1A,E). Treatment with 10, 20 and 50 mg/ml n-LDL for 24 h had minimal effects on b-galactosidase activity compared to untreated control cells (10 mg/ml n-LDL: 5.8 2.7%; 20 mg/ml n-LDL: 7.0 3.6%; 50 mg/ml n-LDL: 9.8 5.3%) (Fig. 1B,E). However, incubation with 10, 20 and 50 mg/ml ox-LDL for 24 h upregulated the number of b-galactosidase positive cells
In a subsequent approach, we investigated the effect of n-LDL, ox-LDL and HDL on the mRNA expression of transforming growth factor-beta2 (TGF-b2). The signals generated by real-time PCR analysis of untreated control cells were set to 1.0 (Fig. 4). Exposure to 10 and 20 mg/ml of n-LDL had no effect on the TGF-b2 mRNA expression (10 mg/ml n-LDL: 1.0 0.02 fold; 20 mg/ml n-LDL: 1.0 0.2 fold), whereas 50 mg/ml of n-LDL increased TGF-b2 mRNA amount by 1.7 0.3 fold compared to untreated control cells (Fig. 4A). Treatment of RPE cells with 10, 20 and 50 mg/ml of ox-LDL for 24 h under serum-free medium conditions increased the mRNA expression of TGF-b2 dose-dependently (10 mg/ml ox-LDL: 1.7 0.2 fold; 20 mg/ml ox-LDL: 2.4 0.4 fold; and 50 mg/ml ox-LDL:
498
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
4. Discussion
Table 1 Primers used for real-time PCR. Gene target
Gene sequence
Gene position
Fibronectin
50 -ctggccgaaaatacattgtaaa-30 50 -ccacagtcgggtcaggag-30
2611–2632 2707–2724
Laminin alpha 1
50 -ctaagctggctcccgatg-30 50 -catccaccacacactgttctg-30
8130–8147 8187–8207
Collagen type IV alpha 2
50 -gggatatttggcctgaaagg-30 50 -caggaagagcagcagaacct-30
4147–4166 4191–4210
TGF-b2
50 -caaagggtacaatgccaactt-30 50 -cagatgcttctggatttatggtatt-30
1195–1215 1283–1307
18S rRNA
50 -ctcaacacgggaaacctcac-30 50 -cgctccaccaactaagaacg-30
1348–1367 1438–1457
4.2 0.4 fold) (Fig. 4A). Treatment of RPE cells with up to 50 mg/ml of HDL did not induce any changes of TGF-b2 mRNA expression compared to untreated control cells (10 mg/ml n-LDL: 1.0 0.1 fold; 20 mg/ml n-LDL: 0.9 0.1 fold; 50 mg/ml n-LDL: 0.8 0.2 fold) (Fig. 4A). To determine total TGF-b2 protein levels released into culture medium after treatment with lipoproteins, a commercially available enzyme-linked immunosorbent assay (ELISA) was used. We found a basal level of 45 8 pg/ml TGF-b2 protein in untreated control cells (Fig. 4B). Treatment of RPE cells with 50 mg/ml n-LDL and 50 mg/ ml ox-LDL increased the TGF-b2 protein secretion to 196 35 pg/ml and 396 32 pg/ml (Fig. 4B). Lower concentrations of n-LDL and oxLDL also increased TGF-b2 protein expression (10 mg/ml n-LDL: 78 5 pg/ml; 20 mg/ml n-LDL: 120 23 pg/ml.; 10 mg/ml ox-LDL: 120 10 pg/ml, 20 mg/ml ox-LDL: 240 38 pg/ml) (Fig. 4B). We could not detect any increase of TGF-b2 protein levels after exposure to HDL (10 mg/ml HDL: 48 6 pg/ml; 20 mg/ml HDL: 44 14 pg/ml; 50 mg/ml HDL: 47 18 pg/ml) (Fig. 4B).
To examine the possible involvement of native low-density lipoprotein (n-LDL), oxidized low-density lipoprotein (ox-LDL) and high-density lipoprotein (HDL) in the pathogenesis of age-related macular degeneration (AMD), we have chosen to study their influence on four representative characteristics of different stages of AMD. In the present study, we demonstrated that n-LDL and more potently ox-LDL induce senescent changes, synthesis of extracellular matrix (ECM) components, cell loss, and expression of transforming growth factor-beta2 (TGF-b2) in the retinal pigment epithelium (RPE) in vitro. Until now, the sequence of cellular events leading to the progression of AMD is still under debate. One theoretical concept implies that senescent changes in the outer retina, the RPE, the Bruch’s membrane and the choroids might be the underlying process for the development of AMD (Roth et al., 2004; Zarbin, 2004). For the pathogenesis of both AMD and arteriosclerosis, it is assumed that increased senescent changes actually progress to the stage of a degenerative disease (Minamino and Komuro, 2007; Young, 1987). Previously, it has been demonstrated that advanced senescence is inducible by oxidative stress in cultured human RPE cells (Honda et al., 2002; Nilsson et al., 2003). In our experiments, ox-LDL promoted advanced senescence of cultured human RPE cells. In contrast, n-LDL and HDL had no effect on advanced senescence. In cultured human endothelial cells, advanced senescence has also been induced by ox-LDL (Imanishi et al., 2004; Maier et al., 1996). This observation is in accordance with endothelial senescence as seen in arteriosclerosis (Minamino and Komuro, 2007; Shi et al., 2007). Whether or not senescent changes in the RPE, as described for AMD (Feher et al., 2006), are induced by ox-LDL is still unknown. Advanced senescence of RPE cells eventually leads to RPE dysfunction, which is majorly manifested by incomplete degradation and increased aggregation of extracellular matrix (ECM)
Fig. 1. RPE cell senescence was evaluated by SA-b-Gal activity after treatment with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Representative photomicrographs show SA-b-Gal positive cells staining blue in RPE cells either (A) untreated or treated with 50 mg/ml (B) n-LDL, (C) ox-LDL or (D) HDL. (E) Quantification of the SA-b-Gal activity assay. The percentage of SA-b-Gal positive cells was scored by counting at least 700 cells in phase-contrast photomicrographs of representative fields. Data (mean s.d.) are based on the sampling of 6–10 photomicrographs per condition in three independent experiments with three different cell cultures from different donors (*P < 0.05). Scale bar: 100 mm.
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
499
Fig. 2. Analysis of lipoprotein effects on mRNA expression of (A) fibronectin (Fn), (B) laminin alpha 1 (Laa1) and (C) collagen type IV alpha 2 (Col4a2) in cultured human RPE cells by real-time PCR. Cells were treated with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Results were normalized to 18S rRNA as reference. Results are given as mean s.d. of three independent experiments with three different cell cultures from different donors (*P < 0.05).
deposits (Young, 1987; Zarbin, 2004). ECM formation belongs to the key pathological factors of AMD and is also known to be inducible by oxidative stress (Roth et al., 2004; Zarbin, 2004). We have demonstrated that both n-LDL and ox-LDL lead to induced expression of ECM components such as fibronectin (Fn), laminin alpha 1 (Laa1) and collagen type IV alpha 2 (Col4a2) in cultured human RPE cells. Previous studies showed that n-LDL and ox-LDL can act profibrogenic by stimulating ECM synthesis in mesangial
cells (Lee, 1999; Roh et al., 1998), human coronary artery smooth muscle cells (Bachem et al., 1999), human glomerular epithelial (Ding et al., 1997) and human hepatic stellate cells (Schneiderhan et al., 2001). The causal relationship between ox-LDL and ECM alterations is supported by their close spatial relation as found in the thickened intima of human coronary arteries (Fukuchi et al., 2002). Whether or not internalized ox-LDL in RPE cells triggers ECM synthesis in vivo is still unclear.
500
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
Fig. 3. Live dead assay of cultured human RPE cells treated with 10–100 mg/ml lipoproteins. Representative fluorescence photomicrographs of nonviable RPE cells show either (A) untreated or treated with 100 mg/ml (B) native (n)-LDL, (C) oxidized (ox)-LDL or (D) HDL. (E–H) Total number of RPE cells in the corresponding fields. (J) Quantification of the effects of lipoprotein treatment on the number of nonviable cells. The percentage of dead cells was scored by counting at least 700 cells in fluorescence photomicrographs of representative fields. Data (mean s.d.) are based on the sampling of 6–10 photomicrographs per condition in three independent experiments with three different cell cultures from different donors (*P < 0.05). Scale bar: 100 mm.
In face of the previous described pathological events such as cellular senescence and ECM formation, deteriorating RPE function may ultimately progress to RPE degeneration with RPE cell death (Young, 1987; Zarbin, 2004). RPE cell death is the manifestation of the most prevalent atrophic form of AMD (Petrukhin, 2007). In our experiments, ox-LDL markedly induced cell death in cultured human RPE cells. A lower cytotoxic effect was observed for n-LDL. Similarly, higher cytotoxic potency of ox-LDL compared to n-LDL has been described in other cellular systems such as human coronary artery smooth muscle cells (Bachem et al., 1999), coronary arterial endothelial cells (Li et al., 1998), and monocytes–macrophages (Hardwick et al., 1996). A number of studies have already demonstrated the cytotoxicity of ox-LDL in cultured human RPE cells with a similar treatment regimen (Gordiyenko et al., 2004; Hoppe et al., 2004; Rodriguez et al., 2004). In the study of Rodriguez et al. (2004), it was assumed that the formation of 7-ketocholesterol, an oxidized lipid in ox-LDL particles, may be responsible for the cytotoxicity of ox-LDL (Rodriguez et al., 2004). Further extended investigations are required to single out the specific component of ox-LDL underlying the cytotoxicity of ox-LDL in human RPE cells. It is still controversial whether and how pathogenic events such as senescent RPE changes, ECM deposition and RPE dysfunction elicit RPE atrophy (‘dry’ AMD) or choroidal neovascularization (‘wet’ AMD) or rather provoke both forms simultaneously (Young, 1987; Zarbin, 2004). One common finding of ‘dry’ and ‘wet’ AMD is the increased expression of TGF-b observed in the RPE cells both of human maculae with ‘dry’ AMD and of surgically removed neovascular membranes from eyes of ‘wet’ AMD (Kliffen et al., 1997). In
‘dry’ AMD, TGF-b has been found to be a promoting factor for ECM formation (Kliffen et al., 1997). Therefore, we have examined the influence of LDL and ox-LDL on TGF-b expression. In our study, both ox-LDL and, in a less marked manner, n-LDL increased TGF-b2 expression in cultured human RPE cells. Similarly, in cultured human endothelial (Maier et al., 1996) or glomerular epithelial cells (Ding et al., 1997), ox-LDL is able to induce increased levels of TGFb2. In contrast, treatment with high-density lipoprotein (HDL) had no effects on TGF-b2 levels in our experiments. To our knowledge, no data are yet available demonstrating the influence of lipoproteins on TGF-b2 production in the cultured human RPE cells. This information could help us to better understand the role of TGF-b2 in the human RPE in vivo. The source and mechanism of deposition of LDL and ox-LDL in the RPE cells are still obscure. On the one hand, there is evidence that de novo synthesis of lipoproteins by the RPE may occur (Fliesler et al., 1993; Malek et al., 2003). On the other hand, an external pathway for plasma lipoprotein accumulation via the fenestrated choroidal cell junctions and Bruch’s membrane into the RPE cells has been suggested in the in vivo situation (Gordiyenko et al., 2004). Concerning cultured human RPE cells, ox-LDL is internalized by CD36 scavenger receptors. With immunohistochemical and real-time PCR analysis we were able to demonstrate the expression of the CD36 scavenger receptor in our primary RPE cells (data not shown). These results are in accordance with a number of studies demonstrating that CD36 scavenger receptors are expressed both in the RPE (Gordiyenko et al., 2004; Ryeom et al., 1996; Yesner et al., 1996) and vascular endothelial cells (Boullier et al., 2001). In the pathogenesis of
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
501
Fig. 4. (A) Analysis of lipoprotein effects on TGF-b2 mRNA expression in cultured human RPE cells by real-time PCR. Cells were treated with 10, 20 and 50 mg/ml native (n)-LDL, oxidized (ox)-LDL and HDL for 24 h under serum-free medium conditions. Results were normalized to 18S rRNA as reference. Results are given as mean s.d. of three independent experiments with three different cell cultures from different donors (*P < 0.05). (B) TGF-b2 protein secretion by RPE cells treated as in (A) and analyzed by ELISA assay. Results expressed as described in (A).
arteriosclerosis, the expression of CD36 scavenger receptors is the major route of internalization of ox-LDL (Boullier et al., 2001). Other well-known ligands for CD36 scavenger receptors are modified lipids of rod outer segment (ROS) membranes (Ryeom et al., 1996). ROS are shed daily from the distal ends of the photoreceptors by RPE cells (Sarangarajan and Apte, 2005). The process of degradation of internalized ROS involves the process of lysosome– phagosome fusion (Sarangarajan and Apte, 2005). CD36 scavenger receptors participate in the recognition and internalization of ROS (Ryeom et al., 1996). ROS membranes, which are rich in long chain polyunsaturated fatty acids, can be modified by lipid peroxidation leading to the creation of oxidized ROS (Hoppe et al., 2004; Ryeom et al., 1996). Since both ox-ROS and ox-LDL are recognized by CD36 scavenger receptors and may perturb the lysosomal function of the RPE (Hoppe et al., 2004; Sarangarajan and Apte, 2005), it is tempting to speculate that the possible involvement of ox-LDL in the induction of AMD-like changes in this in vitro model might actually represent changes that are in vivo caused by ox-ROS. In conclusion, our findings showed that n-LDL and, in a greater extent, ox-LDL induce senescent changes, synthesis of ECM components, cell loss, and expression of TGF-b2 in cultured human RPE cells. In the in vivo situation, these pathogenic cellular events are shared by AMD and atherosclerosis. Our experiments demonstrated that n-LDL and more significantly ox-LDL induce cellular events in cultured human RPE cells, which may resemble pathogenic changes in AMD. Therefore, this study may provide some
fundamental results needed for further investigations on the effects of lipoproteins in the RPE. Acknowledgements The authors thank B. Zimmer, J. Moriniere and K. Obholzer for excellent technical assistance, Harald Kroehn for preparing the figures. This work was supported by DFG WE 2577/2-1 and DOG Research Support to UW-L. References Alge, C.S., Priglinger, S.G., Neubauer, A.S., Kampik, A., Zillig, M., Bloemendal, H., Welge-Lussen, U., 2002. Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin. Invest. Ophthalmol. Vis. Sci. 43, 3575–3582. Al-Shaer, M.H., Choueiri, N.E., Correia, M.L., Sinkey, C.A., Barenz, T.A., Haynes, W.G., 2006. Effects of aging and atherosclerosis on endothelial and vascular smooth muscle function in humans. Int. J. Cardiol. 109, 201–206. An, E., Lu, X., Flippin, J., Devaney, J.M., Halligan, B., Hoffman, E.P., Strunnikova, N., Csaky, K., Hathout, Y., 2006. Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors. J. Proteome Res. 5, 2599–2610. Bachem, M.G., Wendelin, D., Schneiderhan, W., Haug, C., Zorn, U., Gross, H.J., SchmidKotsas, A., Gru¨nert, A., 1999. Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells. Clin. Chem. Lab. Med. 37, 319–326. Boullier, A., Bird, D.A., Chang, M.K., Dennis, E.A., Friedman, P., Gillotre-Taylor, K., Ho¨rkko¨, S., Palinski, W., Quehenberger, O., Shaw, P., Steinberg, D., Terpstra, V.,
502
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503
Witztum, J.L., 2001. Scavenger receptors, oxidized LDL and atherosclerosis. Ann. N. Y. Acad. Sci. 947, 214–222. Campochiaro, P.A., Jerdon, J.A., Glaser, B.M., 1986. The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro. Invest. Ophthalmol. Vis. Sci. 27, 1615–1621. Chamberlain, M., Baird, P., Dirani, M., Guymer, R., 2006. Unraveling a complex genetic disease: age-related macular degeneration. Surv. Ophthalmol. 51, 576–586. Cipollone, F., Fazia, M., Mincione, G., Iezzi, A., Pini, B., Cuccurullo, C., Ucchino, S., Spigonardo, F., Di Nisio, M., Cuccurullo, F., Mezzetti, A., Porreca, E., 2004. Increased expression of transforming growth factor-beta1 as a stabilizing factor in human atherosclerotic plaques. Stroke 35, 2253–2257. DeWan, A., Bracken, M.B., Hoh, J., 2007. Two genetic pathways for age-related macular degeneration. Curr. Opin. Genet. Dev. 17, 228–233. Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U. S. A. 92, 9363–9367. Ding, G., van Goor, H., Ricardo, S.D., Orlowski, J.M., Diamond, J.R., 1997. Oxidized LDL stimulates the expression of TGF-beta and fibronectin in human glomerular epithelial cells. Kidney Int. 51, 147–154. Edwards, A.O., Malek, G., 2007. Molecular genetics of AMD and current animal models. Angiogenesis 10, 119–132. Feher, J., Kovacs, I., Artico, M., Cavallotti, C., Papale, A., Balacco Gabrieli, C., 2006. Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration. Neurobiol. Aging 27, 983–993. Fliesler, S.J., Florman, R., Rapp, L.M., Pittler, S.J., Keller, R.K., 1993. In vivo biosynthesis of cholesterol in the rat retina. FEBS Lett. 335, 234–238. Fukuchi, M., Watanabe, J., Kumagai, K., Baba, S., Shinozaki, T., Miura, M., Kagaya, Y., Shirato, K., 2002. Normal and oxidized low density lipoproteins accumulate deep in physiologically thickened intima of human coronary arteries. Lab. Invest. 82, 1437–1447. Godley, B.F., Jin, G.F., Guo, Y.S., Hurst, J.S., 2002. Bcl-2 overexpression increases survival in human retinal pigment epithelial cells exposed to H2O2. Exp. Eye Res. 74, 663–669. Gordiyenko, N., Campos, M., Lee, J.W., Fariss, R.N., Sztein, J., Rodriguez, I.R., 2004. RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 45, 2822–2829. Guymer, R.H., Chiu, A.W., Lim, L., Baird, P.N., 2005. HMG CoA reductase inhibitors (statins): do they have a role in age-related macular degeneration? Surv. Ophthalmol. 50, 194–206. Hardwick, S.J., Hegyi, L., Clare, K., Law, N.S., Carpenter, K.L., Mitchinson, M.J., Skepper, J.N., 1996. Apoptosis in human monocyte–macrophages exposed to oxidized low density lipoprotein. J. Pathol. 179, 294–302. Hawkins, B.S., Bird, A., Klein, R., West, S.K., 1999. Epidemiology of age-related macular degeneration. Mol. Vis. 5, 26. Holz, F.G., Sheraidah, G., Pauleikhoff, D., Bird, A.C., 1994. Analysis of lipid deposits extracted from human macular and peripheral Bruch’s membrane. Arch. Ophthalmol. 112, 402–406. Honda, S., Hjelmeland, L.M., Handa, J.T., 2002. Senescence associated beta galactosidase activity in human retinal pigment epithelial cells exposed to mild hyperoxia in vitro. Br. J. Ophthalmol. 86, 159–162. Hoppe, G., O’Neil, J., Hoff, H.F., Sears, J., 2004. Accumulation of oxidized lipid– protein complexes alters phagosome maturation in retinal pigment epithelium. Cell Mol. Life Sci. 61, 1664–1674. Hyman, L., Neborsky, R., 2002. Risk factors for age-related macular degeneration: an update. Curr. Opin. Ophthalmol. 13, 171–175. Imanishi, T., Hano, T., Sawamura, T., Nishio, I., 2004. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. Clin. Exp. Pharmacol. Physiol. 31, 407–413. Kamei, M., Yoneda, K., Kume, N., Suzuki, M., Itabe, H., Matsuda, K., Shimaoka, T., Minami, M., Yonehara, S., Kita, T., Kinoshita, S., 2007. Scavenger receptors for oxidized lipoprotein in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 48, 1801–1807. Klein, R., Klein, B.E., Linton, K.L.P., 1992. Prevalence of age-related maculopathy: the Beaver Dam Eye Study. Ophthalmology 99, 933–943. Klein, R., Klein, B.E., Tomany, S.C., Cruickshanks, K.J., 2003. The association of cardiovascular disease with the long-term incidence of age-related maculopathy: the Beaver Dam Eye study. Ophthalmology 110, 636–643. Klein, R., Peto, T., Bird, A., Vannewkirk, M.R., 2004. The epidemiology of age-related macular degeneration. Am. J. Ophthalmol. 137, 486–495. Kliffen, M., Sharma, H.S., Mooy, C.M., Kerkvliet, S., de Jong, P.T., 1997. Increased expression of angiogenic growth factors in age-related maculopathy. Br. J. Ophthalmol. 81, 154–162. Lee, H.S., 1999. Oxidized LDL, glomerular mesangial cells and collagen. Diabetes Res. Clin. Pract. 45, 117–122. van Leeuwen, R., Klaver, C.C., Vingerling, J.R., Hofman, A., de Jong, P.T., 2003. Epidemiology of age-related maculopathy: a review. Eur. J. Epidemiol. 18, 845–854. Leschey, K.H., Hackett, S.F., Singer, J.H., Campochiaro, P.A., 1990. Growth factor responsiveness of human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 31, 839–846. Li, D., Yang, B., Mehta, J.L., 1998. Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas. Am. J. Physiol. 275, 568–576. Maier, J.A., Barenghi, L., Bradamante, S., Pagani, F., 1996. Induction of human endothelial cell growth by mildly oxidized low density lipoprotein. Atherosclerosis 123, 115–121.
Malek, G., Li, C.M., Guidry, C., Medeiros, N.E., Curcio, C.A., 2003. Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with agerelated maculopathy. Am. J. Pathol. 162, 413–425. Mares-Perlman, J.A., Brady, W.E., Klein, R., VandenLangenberg, G.M., Klein, B.E., Palta, M., 1995. Dietary fat and age-related maculopathy. Arch. Ophthalmol. 113, 743–748. Minamino, T., Miyauchi, H., Yoshida, T., Tateno, K., Komuro, I., 2004. The role of vascular cell senescence in atherosclerosis: antisenescence as a novel therapeutic strategy for vascular aging. Curr. Vasc. Pharmacol. 2, 141–148. Minamino, T., Komuro, I., 2007. Vascular cell senescence: contribution to atherosclerosis. Circ. Res. 100, 15–26. Mitchell, P., Wang, J.J., Smith, W., Leeder, S.R., 2002. Smoking and the 5-year incidence of age-related maculopathy: the Blue Mountains Eye study. Arch. Ophthalmol. 120, 1357–1363. Nakajima, K., Nakano, T., Tanaka, A., 2006. The oxidative modification hypothesis of atherosclerosis: the comparison of atherogenic effects on oxidized LDL and remnant lipoproteins in plasma. Clin. Chim. Acta 367, 36–47. Newby, A.C., 2005. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol. Rev. 85, 1–31. Nilsson, S.E., Sundelin, S.P., Wihlmark, U., Brunk, U.T., 2003. Aging of cultured retinal pigment epithelial cells: oxidative reactions, lipofuscin formation and blue light damage. Doc. Ophthalmol. 106, 13–16. Petrukhin, K., 2007. New therapeutic targets in atrophic age-related macular degeneration. Expert Opin. Ther. Targets 11, 625–639. Rahaman, S.O., Lennon, D.J., Febbraio, M., Podrez, E.A., Hazen, S.L., Silverstein, R.L., 2006. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell Metab. 4, 211–221. Robman, L., McNeil, J., Dimitrov, P., Dowrick, A., Tikellis, G., Nicolas, C., Cameron, J., Guymer, R., McGrath, B., McCarty, C., 2004. Methodology of the Cardiovascular Health and Age-Related Maculopathy (CHARM) Study. Ophthalmic Epidemiol. 11, 161–179. Rodriguez, I.R., Alam, S., Lee, J.W., 2004. Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol. Invest. Ophthalmol. Vis. Sci. 45, 2830–2837. Roh, D.D., Kamanna, V.S., Kirschenbaum, M.A., 1998. Oxidative modification of lowdensity lipoprotein enhances mesangial cell protein synthesis and gene expression of extracellular matrix proteins. Am. J. Nephrol. 18, 344–350. Roth, F., Bindewald, A., Holz, F.G., 2004. Keypathophysiologic pathways in agerelated macular disease. Graefe’s Arch. Clin. Exp. Ophthalmol. 242, 710–716. Ruberti, J.W., Curcio, C.A., Millican, C.L., Bailey, T., Kruth, H.S., 2001. Accumulation of cholesterol with age in human Bruch’s membrane. Invest. Ophthalmol. Vis. Sci. 42, 265–274. Rubic, T., Lorenz, R.L., 2006. Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10. Cardiovasc. Res. 69, 527–535. Ryeom, S.W., Silverstein, R.L., Scotto, A., Sparrow, J.R., 1996. Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36. J. Biol. Chem. 271, 20536–20539. Sarangarajan, R., Apte, S.P., 2005. Review melanization and phagocytosis: implications for age related macular degeneration. Mol. Vis. 11, 482–490. van der Schaft, T.L., Mooy, C.M., de Bruijn, W.C., Bosman, F.T., de Jong, P.T., 1994. Immunohistochemical light and electron microscopy of basal laminar deposit. Graefe’s Arch. Clin. Exp. Ophthalmol. 232, 40–46. Schmidt, S., Qin, X., Schmidt, M.A., Martin, E.R., Hauser, E.R., 2007. Interpreting analyses of continuous covariates in affected sibling pair linkage studies. Genet. Epidemiol. 31, 541–552. Schneiderhan, W., Schmid-Kotsas, A., Zhao, J., Gru¨nert, A., Nu¨ssler, A., Weidenbach, H., Menke, A., Schmid, R.M., Adler, G., Bachem, M.G., 2001. Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis. Hepatology 34, 729–737. Schulz, T., Schiffl, H., Scheithe, R., Hrboticky, N., Lorenz, R., 1995. Preserved antioxidative defense of lipoproteins in renal failure and during hemodialysis. Am. J. Kidney Dis. 25, 564–571. Schwartz, C.J., Valente, A.J., Sprague, E.A., Kelley, J.L., Nerem, R.M., 1991. The pathogenesis of atherosclerosis: an overview. Clin. Cardiol. 14, 11–16. Shi, Q., Hubbard, G.B., Kushwaha, R.S., Rainwater, D., Thomas 3rd, C.A., Leland, M.M., Vandeberg, J.L., Wang, X.L., 2007. Endothelial senescence after high-cholesterol, high-fat diet challenge in baboons. Am. J. Physiol. Heart Circ. Physiol. 292, 2913–2920. Sivaprasad, S., Bailey, T.A., Chong, V.N., 2005. Bruch’s membrane and the vascular intima: is there a common basis for age-related changes and disease? Clin. Exp. Ophthalmol. 33, 518–523. Smith, W., Assink, J., Klein, R., Mitchell, P., Klaver, C.C., Klein, B.E., Hofman, A., Jensen, S., Wang, J.J., de Jong, P.T., 2001. Risk factors for age-related macular degeneration: pooled findings from three continents. Ophthalmology 108, 697–704. Stoneman, V.E., Bennett, M.R., 2004. Role of apoptosis in atherosclerosis and its therapeutic implications. Clin. Sci. (Lond.) 107, 343–354. Takahashi, Y., Zhu, H., Yoshimoto, T., 2005. Essential roles of lipoxygenases in LDL oxidation and development of atherosclerosis. Antioxid. Redox Signal 7, 425–431. Tan, J.S., Mitchell, P., Smith, W., Wang, J.J., 2007. Cardiovascular risk factors and the long-term incidence of age-related macular degeneration: the Blue Mountains Eye Study. Ophthalmology 114, 1143–1150. Thornton, J., Edwards, R., Mitchell, P., Harrison, R.A., Buchan, I., Kelly, S.P., 2005. Smoking and age-related macular degeneration: a review of association. Eye 19, 935–944.
A.L. Yu et al. / Experimental Eye Research 88 (2009) 495–503 Tsimikas, S., 2006. Oxidized low-density lipoprotein biomarkers in atherosclerosis. Curr. Atheroscler. Rep. 8, 55–61. Vingerling, J.R., Dielemans, I., Hofman, A., Grobbee, D.E., Hijmering, M., Kramer, C.F., de Jong, P.T., 1995a. The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology 102, 205–210. Vingerling, J.R., Dielemans, I., Bots, M.L., Hofman, A., Grobbee, D.E., de Jong, P.T.V.M., 1995b. Age-related macular degeneration is associated with atherosclerosis: the Rotterdam Study. Am. J. Epidemiol. 142, 404–409. Weber, C., Draude, G., Weber, K.S., Wubert, J., Lorenz, R.L., Weber, P.C., 1999. Downregulation by tumor necrosis factor-alpha of monocyte CCR2 expression and monocyte chemotactic protein-1-induced transendothelial migration is antagonized by oxidized low-density lipoprotein: a potential
503
mechanism of monocyte retention in atherosclerotic lesions. Atherosclerosis 145, 115–123. Yesner, L.M., Huh, H.Y., Pearce, S.F., Silverstein, R.L., 1996. Regulation of monocyte CD36 and thrombospondin-1 expression by soluble mediators. Arterioscler. Thromb. Vasc. Biol. 16, 1019–1025. Young, R.W., 1987. Pathophysiology of age-related macular degeneration. Surv. Ophthalmol. 31, 291–306. Zarbin, M.A., 2004. Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122, 598–614. Zhu, C., Ying, D., Zhou, D., Mi, J., Zhang, W., Chang, Q., Li, L., 2005. Expression of TGFbeta1 in smooth muscle cells regulates endothelial progenitor cells migration and differentiation. J. Surg. Res. 125, 151–156.